Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for uptake of nutrients, hormones and antibodies, for remodeling of plasma membrane (PM) composition in response to environmental changes, and for the regulation of signaling from cell surface receptor tyrosine kinases and G-protein coupled receptors. Thus, CME plays a critical role in many aspects of cellular differentiation, physiology and homeostasis. Consequently, defects in CME impinge on many human diseases, including cancer, cardiovascular disease, diabetes, neurological defects and others. CME is a multistep process initiated by the assembly of clathrin and the AP2 adaptor complex to form nascent clathrin-coated pits (CCPs). Subsequent steps including CCP stabilization, invagination, maturation and their scission from the PM to release clathrin-coated vesicles (CCVs) are orchestrated by myriad endocytic accessory proteins (EAPs). While EAPs have been functionally classified based on domain structure, and the timing of their recruitment to CCPs, in most cases their precise functions remain poorly understood, and in many cases still controversial. Under the auspices of this grant, we have developed accurate and highly sensitive particle tracking software to quantitatively measure the dynamics of clathrin-eGFP labeled CCPs imaged by live cell total internal reflection fluorescence microscopy. We have also developed methods of data analyses that allow us to independently measure multiple discrete stages CCV formation. Through this multi-PI mechanism, we have assembled interdisciplinary expertise in biochemistry, cell and molecular biology, quantitative live cell microscopy, biophysicists, statistics and computational biology. Hence, we are uniquely positioned, as proposed in Aim 1, to conduct the first comprehensive phenotypic characterization of EAPs following CRISPRi knockdown. Completion of these studies will define the stage-specific roles of EAPs in CME. A subset of EAPs chosen based on the severity and novelty of their phenotypes and whether they function at critical nodes for regulation of CME will be analyzed in more detail in Aim 2 to mechanistically dissect their roles in CME. While the functions of few EAPs have been unambiguously defined, nonetheless much is known about the molecular machinery driving CME. Thus, this complex process is ripe for the application of modeling approaches that describe mechanochemical events driving CCV formation based on first principles from physics. Such a model will then serve in the coming years as an integrator of data on the contributions of individual EAPs at discrete stages of CME. Thus, in Aim 3, we will develop a biophysical model of CCP assembly, invagination and maturation, and complement it with a computational framework to directly calibrate unknown model parameters based on the phenotypic information from the data acquired in Aims 1 & 2. Completion of these aims will provide an unprecedented depth of understanding of the molecular and physical mechanisms underlying CME.